Abstract A097: Global pharmacodynamic effects uncovered with AP3 phosphoproteomic profiling of novel WEE1/PKMYT1 inhibitor ACR-2316 reveals the critical importance of PLK1 for ACR-2316’s superior preclinical activity and differentiated mechanism of a

Abstract Introduction: Proper cell cycle progression requires coordinated activation of the cell cycle kinases CDK1, CDK2, and PLK1. WEE1 and PKMYT1 kinases act as negative regulators of CDK1/2 through inhibitory phosphorylation of CDK1/2-Y15 and CDK1-T14. Using Acrivon’sPredictive Precision Proteomics (AP3) Generative Phosphoproteomics platform, we previously identified resistance mechanisms for WEE1 inhibition and demonstrated that resistance could be overcome by balanced co-inhibition of PKMYT1. Here, we present unbiased, global pharmacodynamic (PD) and in vivo analyses evaluating ACR-2316, our clinical stage, potentially first and best-in-class WEE1/PKMYT1 inhibitor rationally designed by AP3 and optimized for superior single agent activity. Methods: ACR-2316 was evaluated in PD and efficacy studies using ovarian and lung xenograft models. The clinical WEE1 inhibitors azenosertib and Debio0123, and the PKMYT1 inhibitor lunresertib were administered in parallel at maximum tolerated/formulable doses. Ovarian cancer xenograft tumors were characterized by AP3 phospho-and pan-proteomic profiling for unbiased analyses of global effects on cell cycle and signaling. Drug-regulated biomarkers were evaluated by xenograft tumors immunostaining. Effects of ACR-2316 and other compounds on cells were evaluated by FACS and Western blot. Results: In ovarian and lung cancer xenograft models, treatment with ACR-2316 uniquely resulted in complete regressions, while treatment with WEE1 inhibitors (azenosertib, Debio0123) or the PKMYT1 inhibitor lunresertib, resulted in only stable disease at maximum tolerated/formulable doses. Using AP3, ACR-2316 was designed to induce potent activation of CDK1, CDK2, and PLK1 to ensure tumor cell death. PD analyses through AP3 profiling of human xenograft tumors treated with ACR-2316, compared to benchmark WEE1 or PKMYT1 inhibitors, revealed stronger activation of CDK1/2 and PLK-1, resulting in dysregulated mitotic signaling. Immunostaining of ACR-2316-treated xenograft tumors demonstrated stronger target engagement, increased phosphorylation of CDK1, CDK2, and PLK1 substrates, decreased WEE1 protein expression, and superior activation of DNA damage response, mitotic, and apoptotic markers compared to single-target WEE1 or PKMYT1 inhibitors. In cells, inhibition of CDK1, CDK2, or PLK1 largely reversed incomplete replication exit, premature mitosis, and apoptosis induced by ACR-2316. Co-inhibition of PLK1 with ACR-2316 restored protein expression of RRM2, a critical regulator of the DNA replication nucleotide pool. Conclusion: ACR-2316 is a potential first- and best-in-class dual inhibitor of WEE1 and PKMYT1, rationally designed using Acrivon’s proprietary AP3 platform to deliver superior single-agent efficacy. Potent activation of PLK1, together with CDK1/2, is essential for the superior activity of ACR-2316. ACR-2316 has shown clinical activity in its ongoing Phase 1b monotherapy trial in solid tumors during dose escalation. Citation Format: Subodh Kumar, Joelle Baddour-Sousounis, YingChun Liu Liu, Mohamed Eldeeb, Jessica Hopkins, Kate Rappard, Calvin Yang, Sam Saliba, Eunice Kwon, Georgia Mitsa, Martina Pasetto, Taronish Dubash, Portia Lombardo, William Dahlberg, Amira Elbakry, Zachary Best, Luka Romero, Maria R. Zabala, Nina Lipjankic, Valentina Siino, Ahmed Youssef, Yan He, Reina Improgo, Anil Prasad, Sibgat Choudhury, Magnus E. Jakobsson, Helen Nilsson, Caroline Wigerup, Lei Shi, Ayesha Murshid, Joon Jung, David Proia, Michail Shipitsin, Kristina Masson, Peter Blume-Jensen. Global pharmacodynamic effects uncovered with AP3 phosphoproteomic profiling of novel WEE1/PKMYT1 inhibitor ACR-2316 reveals the critical importance of PLK1 for ACR-2316’s superior preclinical activity and differentiated mechanism of action [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A097.